The vast numbers of candidate proteins generated from genomics programs are creating enormous opportunities in the biotechnology sector. However, efficient and rapid expression of genes in homologous and heterologous expression systems, and high-level expression and efficient isolation of proteins encoded by such genes are often major bottlenecks. In many circumstances, the practical and/or cost-effective expression of recombinant proteins in amounts large enough to allow for subsequent characterization and evaluation is prohibitive. Expression and isolation of recombinant integral membrane (IM) proteins provide one of many examples.
IM proteins constitute nearly 30% of all open reading frames in fully sequenced eukaryotic genomes. They play central roles in living cells with respect to transport processes, intercellular signaling, and regulation of cell growth. Under native conditions, eukaryotic IM proteins are synthesized and integrated cotranslationally into the membrane of the endoplasmic reticulum (ER) through an aqueous hole known as the translocon (Schatz and Dobberstein, Science, 271(5255):1519-1526, 1996). Eukaryotic membrane proteins are usually expressed at low levels and they are often modified (N- and O-glycosylation, palmitoylation, prenylation, myristolation, GPI-modification, protease cleavage) inside the ER lumen. Within the ER lumen, disulfide bonds also can be formed as part of the folding process by a set of disulfide reductases and oxidases.
In principle, both prokaryotic and eukaryotic expression systems can be used for heterologous expression of eukaryotic IM proteins. A variety of expression hosts have been explored for this purpose, including Escherichia coli, Halobacterium, Lactococcus lactis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, baculovirus-infected Sf9 insect cells, mammalian cell lines transfected stably or transiently by expression vectors, and easily grown small animals. Traditionally, bacterial expression hosts, such as E. coli, have been the preferred platform for heterologous high-level expression of recombinant proteins for biochemical and structural research. The reasons for the popularity of these organisms include culture affordability, ease of genetic manipulation, and high yields of desired product. However, the application of this platform to IM proteins has met with limited success.
At least two complications arise with the production of IM proteins in bacteria. First, unlike soluble proteins, IM proteins must be trafficked to the membrane, involving targeting signals that may not be recognized by the machinery within the bacterial host. Second, even in cases where a signal sequence is discernable, high-level production of membrane proteins in E. coli can competitively exclude production of other vital membrane proteins in the E. coli, leading to toxicity. For this reason, traditional methods of IM protein expression in bacteria have utilized low copy-number plasmids with weak promoters that produce low levels of protein, compensated by extremely large culture volumes (Laage and Langosch, Traffic, 2:99-104, 2001). Alternatively, IM proteins can be purposefully targeted to bacterial inclusion bodies (Kiefer et al., Receptors Channels, 7:109-119, 2000). This process necessitates subsequent renaturation of the desired IM protein from these insoluble deposits, which is fraught with empirical difficulties and limited success rates. Both of these options fundamentally limit the application of traditional bacterial methods to production of high levels of recombinant IM proteins in their native conformations.
While fusion partner proteins have been used to assist in the successful production of soluble recombinant proteins, traditional fusion partners have not been useful in the production of IM proteins (Tucker and Grisshammer, Biochem J., 317:891-899, 1996). None of the currently available fusion proteins (e.g., glutathione-S-transferase, maltose binding protein, or thioredoxin) target the construct to the membrane and facilitate membrane insertion.
Compositions and methods that facilitate the production and isolation of recombinant proteins in heterologous expression systems are needed.